US8597947B2ExpiredUtilityA1
Undifferentiated stem cell culture systems
Est. expiryDec 29, 2024(expired)· nominal 20-yr term from priority
Inventors:Benjamin Reubinoff
C12N 2502/1323C12N 2533/52C12N 2501/115C12N 2533/54C12N 2501/16C12N 5/0606C12N 2501/119C12N 2501/13C12N 2501/155
93
PatentIndex Score
20
Cited by
113
References
20
Claims
Abstract
The present application discloses methods expanding SCs in an undifferentiated state, the methods comprising incubating undifferentiated SCs in suspension within a culture system comprising basic medium and knockout serum replacement (KOSR). The methods may also be applicable for selective spontaneous or directed differentiation of SCs into a selected population of somatic cells from a culture system of SCs in suspension, the method further comprising incubating said undifferentiated SCs in culture system that support respectively, spontaneous or directed differentiation of SCs into the selected population of somatic cells. The present application also discloses a culture system for expansion of stem cells (SCs) comprising a suspension of undifferentiated stem cells within basic medium and knockout serum replacement (KOSR). The methods and culture system of the invention may be used for large scale production of differentiated cells.
Claims
exact text as granted — not AI-modifiedThe invention claimed is:
1. A method of expanding human pluripotent stem cells (SCs), the method comprising
incubating human pluripotent SCs in suspension within a culture system comprising basic medium, a knockout serum replacement (KOSR) and a TGFβ superfamily factor,
wherein the human pluripotent SCs do not undergo differentiation during the expansion.
2. The method of claim 1 , wherein the human pluripotent SCs are derived from human pluripotent SC colonies cultivated on a feeder layer or in a feeder free adherent culture system.
3. The method of claim 1 , wherein the suspension of human pluripotent SCs comprises free floating cells, free floating clusters of cells or free floating aggregates of cells.
4. The method of claim 1 , wherein the basic medium has the following composition:
Concentration in uM
CaCl 3 (anhydrous)
1800
FE(N 3 O) 3 9H 2 O
0.2
KCl
5360
MgCl 3 (anhydrous)
812
NaCl
76000
NaHCO 3
880
NaH 2 PO 4 H 2 O
900
ZnSO 4 7H 2 O
0.67
D-glucose
25000
phenol red
23
MOPS
10000
sodium pyruvate
230
L-alanine
20
L-arginine HCl
400
L-asparagine H 2 O
5
L-cysteine
10
L-glutamine
500
Glycine
400
L-histidine HCl H 2 O
200
L-isoleucine
800
L-leucine
800
L-lysine HCl
5
L-methionine
200
L-phenylalanine
400
L-proline
67
L-serine
400
L-threonine
800
L-tryptophan
80
L-tyrosine
400
L-valine
800
D-Ca pantothenate
8
choline chloride
80
folic acid
8
i-inositol
40
niacinamide
30
pyridoxal HCl
20
riboflavin
1
thiamine HCl
10
vitamin B12
0.2.
5. The method of claim 1 , wherein the culture system further comprises one or more element selected from the group consisting of a member of the FGF family, an extracellular matrix (ECM) component, an antibacterial agent, a non-essential amino acid, a neurotrophin, nicotinamide (NA), a bone morphogenic protein (BMP) antagonist and a serum free medium supplement.
6. The method of claim 5 , wherein the ECM is selected from the group consisting of fibronectin, laminin and gelatin; the antibacterial agent is selected from the group consisting of penicillin and streptomycin; the TGFβ superfamily factor is activin A; the BMP antagonist is selected from the group consisting of noggin, chordin and gremlin; the neurotrophin is selected from the group consisting of BDNF, NT3 and NT4; and the serum free medium supplement is Nutridoma-CS.
7. The method of claim 1 , wherein the human pluripotent SCs comprise human embryonic stem cells (hESCs).
8. The method of claim 1 , wherein the expanding is effected in a bioreactor.
9. A culture system comprising a suspension of human pluripotent stem cells (SCs) in a culture medium comprising a basic medium, knockout serum replacement (KOSR), and a TGFβ superfamily factor.
10. The culture system of claim 9 wherein the suspension of human pluripotent stem cells comprises free floating cells, free floating clusters of cells or free floating aggregates of cells.
11. The culture system of claim 9 , wherein the basic medium has the following composition:
Concentration in uM
CaCl 3 (anhydrous)
1800
FE(N 3 O) 3 9H 2 O
0.2
KCl
5360
MgCl 3 (anhydrous)
812
NaCl
76000
NaHCO 3
880
NaH 2 PO 4 H 2 O
900
ZnSO 4 7H 2 O
0.67
D-glucose
25000
phenol red
23
MOPS
10000
sodium pyruvate
230
L-alanine
20
L-arginine HCl
400
L-asparagine H 2 O
5
L-cysteine
10
L-glutamine
500
Glycine
400
L-histidine HCl H 2 O
200
L-isoleucine
800
L-leucine
800
L-lysine HCl
5
L-methionine
200
L-phenylalanine
400
L-proline
67
L-serine
400
L-threonine
800
L-tryptophan
80
L-tyrosine
400
L-valine
800
D-Ca pantothenate
8
choline chloride
80
folic acid
8
i-inositol
40
niacinamide
30
pyridoxal HCl
20
riboflavin
1
thiamine HCl
10
vitamin B12
0.2.
12. The culture system of claim 9 , further comprising one or more element selected from the group consisting of a member of FGF family, an extracellular matrix (ECM) component, an antibacterial agent, a non-essential amino acid, a neurotrophin, nicotinamide (NA), a bone morphogenic protein (BMP) antagonist and a serum free medium supplement.
13. The culture system of claim 12 , wherein the FGF member is FGF-2, the ECM is selected from the group consisting of fibronectin, laminin and gelatin; the antibacterial agent is selected from the group consisting of penicillin and streptomycin; the TGFβ superfamily factor is activin A; the BMP antagonist is selected from the group consisting of noggin, chordin and gremlin; the neurotrophin is selected from the group consisting of BDNF, NT3, and NT4; and the serum free medium supplement is Nutridoma-CS.
14. The culture system of claim 9 , wherein the human pluripotent SCs are pluripotent, human embryonic SCs (hESCs).
15. The culture system of claim 9 , being free of laminin.
16. The culture system of claim 9 , wherein the human pluripotent SCs are cultured in a bioreactor.
17. The method of claim 1 , wherein the TGFβ superfamily factor is activin A.
18. The method of claim 1 , wherein the culture system further comprises a member of the FGF family.
19. The method of claim 18 , wherein the member of the FGF family factor is FGF-2.
20. A culture system comprising a suspension of human pluripotent stem cells (SCs) within in a culture medium comprising a basic medium, knockout serum replacement (KOSR), a TGFβ superfamily member, and one or more element selected from the group consisting of a member of the FGF family, an extracellular matrix (ECM) component, an antibacterial agent, a non-essential amino acid, a neurotrophin, nicotinamide (NA), a bone morphogenic protein (BMP) antagonist and a serum free medium supplement.Join the waitlist — get patent alerts
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